Figure 3.

Sialidase treatment improves the immunological function of monocyte‐derived dendritic cells (MDDCs). MDDCs were treated with sialidase or left untreated, and then incubated or not (control) with Escherichia coli. (a) The expression of interleukin‐10 (IL‐10), IL‐12, tumour necrosis factor‐α (TNF‐α) and IL‐6 cytokine genes was evaluated by quantitative real‐time PCR in total RNA extracted MDDCs (following sialidase and 1‐hr incubation with E. coli). The mRNA levels of each cytokine are expressed as the permillage (‰) of the expression of the endogenous positive control, β‐actin. Values represent the means of at least six independent assays. Statistical significance (*P < 0·05) refers to the difference between untreated and sialidase‐treated MDDCs following E. coli phagocytosis. (b) Representative images of the nuclear factor‐κB (NF‐κB) transcription factor nuclear translocation. Translocation was assessed by labelling MDDCs (following sialidase and a 15‐min incubation with E. coli) with anti‐NF‐κB p65 (red) and staining the cell nucleus with DAPI (blue). Cells were then fixed and analysed by combining colours through microscopy. At least 600 cells in each condition were analysed. (c) Interferon‐γ (IFN‐γ) gene expression was evaluated by quantitative real‐time PCR in total RNA extracted from a 48 hr co‐culture of MDDCs, (following sialidase and a 1‐hr incubation with E. coli) and autologous T lymphocytes, in a DC : T‐lymphocyte ratio of 1 : 4. The IFN‐γ mRNA levels are expressed as the permillage (‰) of the expression of the endogenous positive control, β‐actin. Values represent the means of at least seven independent assays. Statistical significance (*P < 0·05) refers to the difference between untreated and sialidase‐treated MDDCs following E. coli phagocytosis.